The term "sphingolipids" refers to a group of lipids which are derived from sphingosine. Sphingolipids occur frequently in the cellular membranes of animals, plants and microorganisms. The exact function of sphingolipids in humans remains unknown, but it is clear that this group of compounds is involved in the transmission of electrical signals in the nervous system and in the stabilization of cell membranes. It has also been suggested that glycosphingosines have a function in the immune system: specific glycosphingosines function as receptors for bacterial toxins and possibly also as receptors for bacteria and viruses.
Sphingolipids contain sphingosine, dihydrosphingosine or phytosphingosine as a base in amide linkage with a fatty acid. Sphingosine or phytosphingosine bases may be used as starting materials in the synthesis of a particular group of sphingolipids, namely ceramides. Ceramides are the main lipid component of the stratum corneum, the upper layer of the skin. The stratum corneum has an important barrier function, external compounds are generally kept outside of its barrier and the loss of moisture is limited. The addition of sphingolipids such as ceramides to skin cosmetic products improve the barrier function and moisture-retaining properties of the skin (Curatolo, 1987; Kerscher et al., 1991).
Currently, heterogenous sphingolipid preparations for cosmetics are mainly extracted from animal sources. Obviously, this is a rather costly process on an industrial scale. Moreover, it has been found that these materials are potentially unsafe due, for example, to the possible presence of bovine spongiform encephalomyelitis (BSE) in bovine tissue. Thus, the cosmetic industry has demonstrated an increasing interest in new sources of pure, well-defined sphingolipids, which are obtained from sources other than animal tissues.
Microorganisms such as the yeasts Pichia ciferrii, formerly indicated as Hansenula ciferrii and Endomycopsis ciferrii (Barnett et al., 1990; Stodola and Wickerham, 1960; Wickerham and Stodola, 1960; Wickerham et al., 1954; Wickerham, 1951) have been found to produce sphingolipids as such, as well as sphingosine, phytosphingosine and/or derivatives thereof. This discovery provides sources for sphingolipids themselves and for starting materials for the production of other commercially valuable compounds which could offer a viable alternative to the use of animal sources of these compounds.
For example, acetylated derivatives of sphingosine, dihydrosphingosine and phytosphingosine may be deacetylated and the thus-obtained sphingosine, dihydrosphingosine or phytosphingosine may be chemically converted into related compounds such as ceramides, pseudoceramides and/or glycoceramides which in turn may be applied in cosmetic and therapeutic products (Smeets and Weber, 1993).
The production of phytosphingosine and/or its acetylated derivatives has also been demonstrated in the yeasts Candida utilis and Saccharomyces cerevisiae (Wagner and Zofcsik, 1966; Oda and Kamiya, 1958), Hanseniaspora valbvensis (Braun and Snell, 1967) and Torulopsis utilis (Kulmacz and Schroepfer Jr., 1978). Phytosphingosine production has also been reported in the fungi Aspergillus sydowi and Penicillium notatum (Stodola and Wickerham, 1960).
Furthermore, in a study in which thirty species of yeast selected from the genera Saccharomyces, Kluyveromyces, Debaromyces, Pichia, Hansenula, Lipomyces, Sporobolomyces, Cryptococcus, Torulopsis, Candida, Trichosporon and Rhodotorula were examined, it was found that all contained at least a form of sphingolipids (ceramide monohexoside) and thus could potentially be employed for sphingolipid production (Kaneko et al., 1977). In an ethanolamine-producing mutant of Saccharomyces cerevisiae, phytosphingosine was shown to accumulate, thus providing this yeast with a source of ethanolamine (Ishida-Schick and Atkinson, 1983).
Sphingolipid production has also been demonstrated in strains of bacterial genera such as Sphingobacterium (Yano et al., 1983), Acetobacter, Bacteroides, Bdellovibrio, Xanthomonas and Flavobacterium (Tahara et al., 1986).
Stoffel et al. (1968) found that the yeast Hansenula (Pichia) ciferrii acetylates all of the long-chain bases which were used as precursors in the study. Sphingosine was converted into triacetylsphingosine and dihydrosphingosine into triacetyl-, diacetyl- and N-acetyl-dihydrosphingosine. Moreover, three acetyl derivatives of phytosphingosine have been isolated, namely tetraacetyl-, triacetyl- and N-acetyl-phytosphingosine. In addition to these acetyl derivatives, Hansenula ciferrii produced long chain ceramides in a medium containing long chain bases.
The biosynthetic pathway of tetraacetylphytosphingosine (TAPS) synthesis in Pichia ciferrii was described by Barenholz et al (1973). The biosynthetic pathway for sphingosine and dihydrosphingosine is proposed by Dimari et al. (1971).
Barenholz et al. (1971 & 1973) investigated the metabolic background of the production of TAPS and other sphingolipid bases in four strains of Hansenula (Pichia) ciferrii. In the later study, the profiles of four microsomal enzymes specific for the biosynthesis of acetylated sphingosine bases of a low (Hansenula ciferrii NRRL Y-1031, E-11, sex b, 8-20-57) and a high producer (Hansenula ciferrii NRRL Y-1031, F-60-10) were compared. It was found that the specific activity of 3-keto dihydrosphingosine synthetase and the long-chain base acetyl-CoA acetyltransferase were increased 5-10 fold and 30 fold respectively, as compared with the low producer, whereas the activities of palmityl thiokinase and 3-ketodihydrosphingosine reductase were similar. This indicates that in the low producer, the activity of the 3-ketodihydrosphingosine synthetase and the long-chain base acetyl-CoA acetyltransferase are the limiting steps in the synthesis of acetylated sphingosines. Under the defined growth conditions, Hansenula ciferrii NRRL Y-1031 F-60-10 was found to produce 300 .mu.moles/1 sphingosine (about 0.15 g/1) bases, of which, at least 250 .mu.moles/1 were extracellular. Even where culture conditions were optimized for TAPS production, only 0.485 g/l TAPS (0.024 g TAPS/ g dry yeast) was obtained (Maister et al., 1962).
However, none of the yeast strains studied to date, even Pichia ciferrii NRRL Y-1031 F-60-10, produce sufficient amounts of sphingolipid bases such as sphingosine, phytosphingosine or derivatives thereof to be an efficient, economically attractive source of such compounds. For example, the availability of yeast strains capable of producing increased levels of TAPS would considerably improve the economic feasibility and attractiveness for the production of this important starting material which in turn may be converted into commercially valuable end-products such as ceramides, pseudoceramides and glycoceramides.